There is an increasing need to find new molecules which can effectively modulate a wide range of biological processes, for applications in medicine and agriculture. A standard way for searching for novel bioactive chemicals is to screen collections of natural materials, such as fermentation broths of plant extracts, or libraries of synthesized molecules using assays which can range in complexity from simple binding reactions to elaborate physiological preparations. The screens often only provide leads which then require further improvement either by empirical methods or by chemical design. The process is time-consuming and costly, but it is unlikely to be totally replaced by rational methods even when they are based on detailed knowledge of the chemical structure of the target molecules. Thus, what we might call "irrational drug design"--the process of selecting the right molecules from large ensembles or repertoires--requires continual improvement both in the generation of repertoires and in the methods of their selection.
Recently, there have been several developments in using peptides or nucleotides to provide libraries of compounds for lead discovery. The methods were originally developed to speed up the determination of epitopes recognized by monoclonal antibodies. For example, the standard serial process of stepwise search of synthetic peptides now encompasses a variety of complex methods in which large arrays of peptides are synthesized in parallel and screened with acceptor molecules labelled with fluorescent or other reporter groups. The sequence of an effective peptide can be decoded from its address in the array. See for example Geysen et al., Proc. Natl. Acad. Sci. USA, 81: 3998-4002 (1984); Maeji et al., J. Immunol. Methods, 146: 83-90 (1992); and Fodor et al., Science, 251: 767-775 (1991).
In another approach, Lam et al., Nature, 354: 82-84 (1991) describe combinatorial libraries of peptides that are synthesized on resin beads such that each resin bead contains about 20 pmoles of the same peptide. The beads are screened with labelled acceptor molecules and those with bound acceptor are searched for by visual inspection, physically removed, and the peptide identified by direct sequence analysis. This method requires, however, sensitive methods for sequence determination.
A different approach for identification in a combinatorial peptide library is used by Houghten et al., Nature, 354: 84-86 (1991). For hexapeptides of the twenty natural amino acids, four hundred separate libraries are synthesized, each with the first two amino acids fixed and the remaining four positions occupied by all possible combinations. An assay, based on competition for binding or other activity, is then used to find the library with an active peptide. Then twenty new libraries are synthesized and assayed to determine the effective amino acid in the third position, and the process is repeated until all six positions in the peptide are identified.
More recently, Houghten (Abstract, European Peptide Society 1992 symposium, Interlaken, Switzerland) suggested a different approach. Starting with twenty amino acids, a total of 20.times.6=120 peptide mixtures are synthesized. In twenty mixtures, position 6 contains a unique amino acid, and positions 1-5 contain a mixture of all natural amino acids. In another twenty mixtures, position 5 contains a unique amino acid and all other positions contain a mixture of all twenty amino acids, etc. Once synthesized, all the 120 peptide mixtures are tested simultaneously and the most active of each of the twenty mixtures representing each position is identified.
A biological method has recently been described in which a library of peptides is presented on the surface of a bacteriophage such that each phage contains a DNA sequence that codes for an individual peptide. The library is made by synthesizing a large number of random oligonucleotides to generate all combinations, followed by their insertion into a phage vector. Each of the sequences is cloned in one phage and the relevant peptide can be selected by finding those that bind to the particular target (by a method known as biopanning). The phages recovered in this way can be amplified and the selection repeated. The sequence of the peptide is decoded by DNA sequencing. See for example Cwirla et al., Proc. Natl. Acad. Sci. USA, 87: 6378-6382 (1990); Scott et al., Science, 249: 386-390 (1990); and Devlin et al., Science, 249: 404-406 (1990).
These libraries may encompass a very large number of different peptides which represent potential ligands to a variety of macromolecules such as receptors, polypeptides, enzymes, carbohydrates and antibodies. Therefore, phage display technology appears to be a very powerful tool for the selection of peptide sequences that bind to a target molecule. These peptides may find numerous applications, namely as antigens in vaccine composition, as enzyme inhibitors, as antagonists or agonists to receptors, for example.
For example, the monoclonal antibody 1B7, first described by Sato et al. (Infect. Immun., 46: 422-428 (1984)) has been raised against the Bordetella pertussis toxin (PTX). This antibody is able to neutralize the toxin in vitro and to protect mice from intracerebral challenge with virulent B. pertussis. The epitope recognized by 1B7 was shown to be discontinuous and largely dependent on conformation. Hoping to obtain peptide sequences that would mimic such a discontinuous epitope, Felici et al. (Gene, 128: 21-27(1993)) constructed two phage display libraries consisting of nine random amino acids inserted in the major coat protein (pVIII), which nanopeptides were linear or flanked by two cysteine residues (circular). The two libraries were screened with the antibody 1B7. The positive clones were sequenced and a consensus sequence was obtained only for linear peptides. In the absence of a three-dimensional structure of the PTX, it is very difficult to determine how the consensus peptide sequence corresponds to amino acid residues of the original protein that are important in the constitution of the discontinuous epitope recognized by the antibody 1B7. Despite this lack of information, the authors expected that the selected nanopeptides would mimic the binding site of the original protein sufficiently to serve as antigens in the production of vaccines against PTX. This was not the case. Indeed, the peptides were able to compete with PTX for the binding site of 1B7, but they were not capable of sufficiently mimicking the discontinuous epitope of PTX to elicit the production of antibodies specific to the original antigen, PTX. Moreover, it is believed that the phage recombinant peptides adopt a conformation that may be governed by the surrounding phage sequences, which conformation is recognizable by 1B7. When peptides alone were synthesized without the presence of surrounding phage sequences, they lost their ability to bind 1B7. It is suggested that more sophisticated or longer peptides might be constructed and might be more successful in mimicking the original epitope.
The same group of researchers (Luzzago et al., Gene, 128:51-57 (1993)) used the same libraries to select oligopeptides that would bind another antibody, H107, that recognizes the native conformation of the recombinant human H-subunit ferritin (H-Fer). This time, the three-dimensional structure of H-Fer was known. The consensus peptide sequence obtained only for the linear selected peptides was used to assign to amino acids, that were space located in the original protein, a putative role in the conformation of the H-Fer epitope. When the peptides were synthesized in the presence of surrounding sequences located in the original protein as well as those synthesized with surrounding phage sequences, the peptides screened with H107 antibody were capable of mimicking the original proteic assembly and bound H107. Accordingly, the different results obtained with peptides selected with two different antibodies suggest that the same libraries were not successful in the selection of epitopes of all existing antigens.
A different shorter peptide library has been obtained by O'Neil et al. (Proteins: Structure, Function and Genetics, 14: 509-515 (1992)). These authors have constructed a random circular hexapeptide sequence inserted in the pIII phage protein. This time, the library was used to select ligands to the receptor glycoprotein IIb/IIIa, a member of the integrin family of cell adhesion molecules that mediate platelet aggregation through the binding of fibrinogen and von Willebrand factor. The purpose of this work was to find ligands useful as antagonists or as antithrombotic agents. The glycoprotein IIb/IIIa binds to a very short sequence commonly known as the RGD sequence. These authors identified a consensus sequence when using a circular library, and identified certain ligands that were better antagonists than the ligand SK106760 (a cyclic peptide developed after extensive array of peptides) used to elute the phages of interest. They also found that a variant RGD sequence wherein the arginine was replaced by a lysine was a strong anti-aggregatory peptide.
Another "genetic" method has been described where the libraries are the synthetic oligonucleotides themselves wherein active oligonucleotide molecules are selected by binding to an acceptor site and are then amplified by the polymerase chain reaction (PCR). PCR allows serial enrichment, and the structure of the active molecules is then decoded by DNA sequencing of clones generated from the PCR products. The repertoire is limited to nucleotides and the natural pyrimidine and purine bases or those modifications that preserve specific Watson-Crick pairing and can be copied by polymerase.
The main advantage of the genetic methods resides in the capacity for cloning and amplification of DNA sequences, which allows enrichment by serial selection and provides a simple and easy method for decoding the structure of active molecules. The prior art results show little success in selecting peptides for a specific molecule, since the length and the conformation of the exposed peptide may be sufficient to retrieve a peptide binding to a specific molecule while it might not be suited to retrieve a peptide that efficiently mimics a more sophisticated binding region on another molecule.
There is therefore a need for other libraries that contain novel peptide sequences at the surface of filamentous phages, which libraries may be used to select ligands to known and unknown molecules. There is also a need for more rational approach to construct libraries suitable for retrieving the best diversity of peptides binding to any ligand.
Furthermore, to date there has been no use of this technology to find peptides that are capable of binding to micromolecules and of mimicking a natural receptor site, and to use these peptides as is or as a second ligand to re-screen libraries to find a new image of the receptor, or as an immunogen to produce antibodies which are another class of effector molecules.